Column tip processing device and column tip processing method

ABSTRACT

The present invention relates to a column tip processing device and a column tip processing method, and its objective is to enhance the contact between a packing and a fluid as a subject to be processed whereby accomplishing a highly efficient reaction as well as an accurate processing, which has a nozzle head having a single or multiple-channeled nozzle, a suction and discharge mechanism, one or more types of column tips in which a packing is enclosed, a stage provided with a fluid housing part group including a plurality of fluid housing parts in which various solutions are or can be housed, and moving means for moving the nozzle head relatively to the fluid housing part group, and also has a controlling part which controls the suction and discharge mechanism and the moving means with regard to quantities, pressure, flow rate, the number of cycles, time or position of the suction or discharge by the nozzle based on a structural requirement relating to the structure of the one or more types of column tips to be fitted to the nozzle and a processing requirement relating to the processing contents involving one or more types of subject fluids subjected to the suction or discharge of the column tips.

TECHNICAL FIELD

The invention relates to a column tip processing device and a column tipprocessing method.

BACKGROUND OF THE INVENTION

There is a liquid chromatograph using a principle of a liquidchromatography in which, to a cylindrical vessel having liquid inlet andoutlet in the center of top and bottom disks referred to as a column, aparticulate packing having 10 to several hundred microns in particlesize referred to as a gel is plugged without allowing for any voids inthe space sandwiched between the top and bottom disks intending toachieve a uniform and sufficient contact with the liquids, and thenseparation and purification of a substance, mainly of a protein, isconducted utilizing the interaction between a solute molecule and thegel upon allowing the liquid to run in a single direction using a pumpand the like from any direction from the liquid inlet or outlet on thetop or bottom. Currently, a gel for a liquid chromatography having avarying surface structure is marketed. One having an ion exchange groupon the surface of a gel has a function as an ion exchange gel, and makesit possible to separate substances by an ion exchange chromatographyutilizing an interaction between a solute molecule and an ionic charge.

For example, when using a liquid chromatography, the separation wasbased on the specificity or the nature of a protein due to a change inthe concentration resulting from mixing of two or three solutions. Insuch a method, a packing is plugged in a column and equilibrated with abuffer with an affinity allowing an intended protein to be adsorbed, andthen a sample protein solution dissolved in the same buffer is passedthrough the column to accomplish the adsorption onto the packing, towhich then a buffer solution having a different concentration is appliedfrom the top of the column using a pump and the like, wherebyestablishing a concentration gradient in the column. At the same time,the concentration gradient in the column is established, and the proteinloses an affinity at a concentration corresponding to the affinity ofthe intended protein, whereby being released from the packing andeluted. Accordingly, when the concentration of the intended protein isunknown, a buffer system allowing for a concentration gradient over awide range is employed, and after fractionation to a plurality of wellsbased on the concentration gradient, the absorbance is measured forexample by a UV absorption detector to narrow the range, and thereaftera buffer system giving a narrow range of the concentration gradient isemployed, whereby extracting the intended protein.

Nevertheless, such a conventional liquid chromatography requiresexpensive devices and buffer solutions as well as atrial-and-error-based and well-trained operation, and suffers fromeffects of the concentration and the flow rate of a developing bufferwhich is not negligible.

Moreover, in such a conventional column, since a liquid is allowed torun in a single direction in a particulate packing plugged in thecolumn, the packing is plugged without allowing for any voids in thecolumn to ensure a high probability of the contact of the packing with aliquid upon passing through it, resulting in the time period of thecontact of the liquid with the packing which can be controlled only bythe flow rate in the column. However, because of the control of the flowrate using a pump, a transporting medium for moving a liquid containingthe intended substance as a processing subject will be required. Thus, abuffer solution will be required in a problematically large quantitywhich is several times, several ten times, several hundred times as thatof the liquid, while the contact with the packing surface will notnecessarily high.

In addition, in a conventional column, since a fluid runs only in asingle direction and the packing is plugged without any void in a spacesandwiched between the disks of the column, a certain fixed routethrough which the liquid runs in the packing tends to be established.Once such a fixed route is established, a part which can not participatein the reaction may problematically occur.

Also since the mobile phase flow rate or mobile phase concentration hasmore or less influence, a troublesome sophisticated control is required.

[Patent Document 1] EP No. 198413 [Patent Document 2] InternationalPublication No. 88/09201 [Non-Patent Document 1] Liquid ChromatographyQ&A, Itaru Matsushita, GIHODO SHUPPAN Co., Ltd., June, 2000 [Non-PatentDocument 2] Practice in Liquid Chromatography, Akira Eto, SANKYO Co.,Ltd., 1976 DISCLOSURE OF THE INVENTION Problems to be Solved by theInvention

Accordingly, the first object of the invention is to provide a columntip processing device and a column tip processing method enabling ahighly efficient reaction, thus a high separation performance whichleads to a rapid processing, by enhancing the contact between a packingand a liquid as a subject to be processed. The second object of theinvention is to provide a column tip processing device and a column tipprocessing method capable of being conducted with no need of a largequantity of transporting medium, thus being conducted with asmall-scaled device. The third object of the invention is to provide acolumn tip processing device and a column tip processing method enablingan automatic processing of column tips continuously using a singledevice. The fourth object of the invention is to provide a column tipprocessing device and a column tip processing method enabling a highlyaccurate control of a liquid chromatography.

Means for Solving the Problems

A first aspect of the present invention is a column tip processingdevice including: a nozzle head having a single or multiple-channelednozzle; a suction and discharge mechanism conducting suction ordischarge of a gas via the nozzle; one or more types of column tips eachhaving a tip-shaped vessel having a fitting opening to be fitted to thenozzle and a hollow end through which a fluid can flow in or out inresponse to the suction or discharge of the gas and a packing enclosedin the tip-shaped vessel; a stage provided with a fluid housing partgroup including a plurality of fluid housing parts into each of whichthe hollow end can be inserted and which house or can house varioussolutions; and moving means for moving the nozzle head relatively to thefluid housing part group; and also having a controlling part whichcontrols the suction and discharge mechanism and the moving means withregard to quantities, pressure, flow rate, the number of cycles, time orposition of suction or discharge by the nozzle based on a structuralrequirement relating to the structure of the one or more types of columntips to be fitted to the nozzle and a processing requirement relating tothe processing contents involving one or more types of subject fluidssubjected to the suction or discharge of the column tips.

As used herein, the term “packing” refers to an insoluble stationaryphase used to adsorb or capture, or react with or bind to a biologicalsubstance contained in a subject fluid which is a subject for suction ordischarge as a so-called mobile phase. Such a packing may include forexample a certain particulate carrier on which surface a biologicalsubstance is placed or bound.

A “carrier” is an insoluble solid capable of adsorbing, reacting with,binding to, immobilizing or capturing a biological substance in a fluid,whose shape may include a plurality of solids such as a plurality ofparticles, powders, blocks and the like. A single particle may also bein the forms not only of spherical forms but also of columns, cylinders,irregular forms. The carrier may have a varying size, such as oneallowing passage through the hollow end and being larger than the poresize of the filter discussed later here. The material for a carrier mayfor example be a gel, porous material, continuous pore-carryingmaterial, hydrous material and the like made from fibers and resins suchas rubbers, silicones, celluloses, nylons and the like, as well asmetals such as non-magnetic particles, magnetic particles and the like.Such a carrier is provided with a chemical substance or a biologicalsubstance such as functional groups for the purpose of adsorption,reaction, binding, immobilization or capture of the biologicalsubstance. Also provided on the surface of the carrier may for examplebe antigens, antibodies, enzymes, substrates, receptors, affinityligands such as His-tag, affinity tags and the like.

A “biological substance” may for example be a biopolymer or smallmolecule such as a genetic substance such as a nucleic acid, protein,saccharide, saccharide chain, peptide, and color. The biologicalsubstance may include a cell, virus, plasmid and the like. Such abiological substance may be employed as a detector which detects,captures, separates and extracts the binding of a biological substanceas a receptor having an ability of binding to such a biologicalsubstance as a ligand. The receptor may for example be a geneticsubstance such as a nucleic acid, protein, saccharide and peptide asdescribed above. A “filter” is a member for filtration or separation ina form of a thin film or thin sheet having a certain pore size, and,when provided in a manner to partition a fluid path, it prevents thepassage of a substance larger than the pore size.

The phrase “absorb or capture the biological substance, or react with orbind to the biological substance” means that a reaction or binding isdue to a covalent bond, chemical adsorption as well as physicaladsorption or electrical interaction or capture in a pore of a certainsize, or a specific reaction with a binding substance in which a certainchemical substance is adsorbed chemically or physically onto orimmobilized on the carrier, and also due to other manners. The carriermay be formed also from a porous material, irregular surface material,fibrous material and the like, whereby enhancing the ability of reactingwith or binding to various substances including biological substances.In order to immobilize complementary biological materials for thereaction with or binding to a biological substance, the carrier isallowed to express or generate a functional group. For this purpose, byhydrolyzing a peptide bond possessed by polyamide-based polymer”-derivedsilks, nylons (3-nylon, 6-nylon, 6,6-nylon, 6,10-nylon, 7-nylon,12-nylon and the like), all aromatic polyamides such aspolyparaphenylene terephthalamide (PPTA), heterocycle-containingaromatic polymers, a functional group used for immobilizing a biologicalsubstance is expressed or generated. Such a functional group capable ofbinding to a biological substance may for example be a carboxyl group(—COOH), an amino group (—NH2), or derivative thereof. The size of apore suitable for immobilizing a biological substance may for example beseveral micrometers or less.

The phrase “to be fitted to a nozzle” includes a direct fitting to thenozzle and a fitting to a member to be fitted to the nozzle, such astip, adapter and the like. The “tip” has a tube having a large diameterand a tube having a small diameter which is formed in fitting with thetube having a large diameter and which is formed in a size smaller thanthat of the tube having a large diameter, and the tube having a largediameter has a fitting opening to be fitted to the nozzle, and the tubehaving a small diameter has a hollow end through which a fluid can flowin or out in response to the suction or discharge of a gas.

A “tip-shaped vessel” is a vessel which has a fitting opening fitted orcapable of being fitted to a member employed for the suction ordischarge and in which a carrier can be housed. Although the tip-shapedvessel can have a thick tube and a narrow tube, there is no limitationto one having a typical tip shape such as a tube having a large diameterand a tube having a small diameter. In such a case, the narrow tube canhave a hollow end and the thick tube can have a fitting opening on theupper side thereof. For example, the thick tube may have a form of aquadratic prism instead of a tube having a large diameter, and thenarrow tube may be a pipe having a polygonal section instead of a tubehaving a small diameter. The thick tube may also be formed as a diskprotruding in the direction of radius relative to the axial directionwhereby reducing the length along with the axis. The packing is housedin a part corresponding to the thick part or a part corresponding to thetransfer part between the thick tube and the narrow tube. In such acase, the stage provided on the part corresponding to the thick tube andanother stage, at a distance from the stage, in the part correspondingto the transfer part may be utilized to partition the filters, and thepacking may be sealed such that the packing is sandwiched between thefilters. The capacity of the tip-shaped vessel is preferably one capableof handling a fluid in a quantity for example from several μ liters toseveral hundred μ liters. In the tip-shaped vessel, a reservoir tubewhich reserves a fluid introduced from the hollow end in addition to thepart housing the packing as being enclosed may also be provided. Such areservoir tube may be formed in a size larger than that of the narrowtube or than that of the thick tube. The narrow tube may be formed asbeing integrated with the thick tube or with the reservoir tube, or maybe detachably formed therefrom. The thick tube itself, and the thicktube and the reservoir tube may be formed integrally or detachably.

The material for a tip-shaped vessel may be a light transmissiblematerial for enabling an optical measurement. The material for thetip-shaped vessel may for example be a resin such as a polyethylene,polypropylene, polystyrene, acrylic material and the like, as well as aglass, metal, metallic compound and the like. The size may for examplebe one allowing several μ liters to several hundred μ liters to behoused in the narrow tube.

A “subject fluid” is a liquid serving as a subject to be suctioned to ordischarged from the column tip, and may for example be a liquidcontaining an intended biological substance, such as a test samplecontaining a substance extracted and separated from a patient, studysubject, animal and others, including a serum, whole blood and the like,and also includes a certain protein, or a series of buffers having astepwise gradient of concentrations having a certain difference inconcentration from each other which is housed in each fluid housing partdescribed above, or various washing solutions employed for washing thecolumn tips, eluents for eluting an intended substance, once adsorbedonto, captured by, reacted with or bound to the packing, from thispacking.

The “structural requirement” is a requirement relating to the structureof a column tip required for setting with regard to suction ordischarge, while the “processing requirement” is a requirement relatingto the processing conducted by a column tip involving a subject fluid tobe the suctioned and discharged. The structural requirement includes aplurality of items since a column tip having a relatively complicatedstructure is employed. The processing requirement also includes aplurality of items relating to the subject fluid.

The phrase “based on the structural requirement and the processingrequirement, . . . quantities, pressure, flow rate, the number ofcycles, time or position of the suction or discharge by the nozzle arecontrolled” is based on various factors serving as a resistance againstthe suction or discharge of a liquid such as filters, mesh platespresent in the column tip as well as the presence of a plurality ofstages provided in the tip-shaped vessel. Accordingly, for the purposeof conducting suction or discharge at a high accuracy, an appropriatesuction or discharge control is performed, upon controlling suction ordischarge utilizing a difference in the pressure of air which is anelastic fluid, while correcting the quantity, time and speed of suctionor discharge based on the structural requirement and the processingrequirement. For example, when the adsorption performance of the packingis lower than a required adsorption performance, then the time periodfor the contact is prolonged to promote the adsorption, resulting in arequirement of repetitive passages through the packing, which leads toincreasing the number of suction and discharge cycles. In addition, insuch a case a relatively slow suction serves to ensure the prevention ofthe detachment of an intended biological substance once adsorbed. In thecase of a higher adsorption performance, it may be highly efficient toestablish a suspending state with the packing by suctioning at a highspeed.

Furthermore, it is also required to correct quantities, time, speed orpressure of the suction or discharge by taking the nature of the subjectfluid as a subject of suction or discharge such as viscosity,temperature, quantity and processing contents into account based on theprocessing requirement. In addition, depending on the ratio between thepacking and the column tip capacity included in the structuralrequirement, the quantity of the fluid capable of being suctioned islimited in order to prevent introduction of air. As used herein, theterm “time” includes a time point and a timing. The term “position”includes the vertical position of the suction or discharge, in additionto the housing position for the subject fluid to be suctioned anddischarged.

The suction and discharge is not necessarily limited to an actualsuction and discharge of a liquid, and may be conducted based on thedetails of each step of such a processing.

For example, in a state where column tips are kept and when the packingactivation maintaining fluid is housed in contact with the packing, thenfor the purpose of avoiding a phenomenon that the air layer in theopening becomes positive correspondingly to the nozzle volume uponnozzle fitting which prevents the fluid in a column tip to be splashedfrom the hollow end of the column tip which leads to a contamination, asuctioning action should be taken correspondingly to the nozzle volumeupon fitting.

Also when transporting column tips by nozzle, a slight suction isconducted to avoid the leakage from the hollow end.

A second aspect of the invention is a column tip processing device,wherein the packing is enclosed in the tip-shaped vessel using at leastone filter provided in a manner to partition the tip-shaped vessel, thestructural requirement includes a plurality of items relating to thestructure of the tip-shaped vessel, the structure of the filter or themorphology, type and nature of the packing enclosed, and the processingrequirement includes a plurality of items relating to the processingcontents including each housing position, type, nature or quantity ofone or more types of the subject fluids subjected to the suction ordischarge of the column tips.

The phrase “the structure of the member (fitted to the nozzle or thetip-shaped vessel” includes, for example, the positions of one or morenozzles having the tips as being fitted thereto, and the morphology andthe size of a tip-shaped vessel. The term “the structure of a filter”includes the filter's pore size, pore density, thickness, area throughwhich a fluid can pass, porosity, and the number of filters. The phrase“the morphology or nature of the packing enclosed” includes, for examplewhen the packing is formed from a particulate carrier, the particlesize, and the ratio of the volume of all particles to the capacity oravailable capacity of the column tip. The term “nature” includes thematerial employed for the packing, the degree of the adsorption,capture, reaction and binding performances of the packing for adsorbing,capturing, reacting with, binding to an intended biological substance.The phrase “nature of the subject fluid” includes the concentration of abiological substance contained in the subject fluid, the viscosity ofthe subject fluid and the like. When the rigidity of the filter is lowand a mesh plate for supporting the filter is provided, then the poresize and the thickness of the mesh plate should also be taken intoaccount.

Also by varying the ratio between the bead volume forming the packingand the tip-shaped vessel's capacity, and especially by selecting thepacking volume which gives a ratio which allows the interstice insteadof a densely enclosed state in the tip-shaped vessel whereby allowingsuction or discharge to be conducted at a flow rate allowing a liquidturbulence to occur in the column tip, a sufficient contact between thepacking and the subject fluid is ensured, whereby enabling a highlyefficient processing. In addition, by taking the pore size or the numberof pores per part area into account, an appropriate suction or dischargeflow rate can be determined.

The phrase “processing contents” includes the types of one or more typesof column tips to be employed, and a protocol showing how to processother tips to be employed in order to achieve the purpose of theprocessing for two or more s of the subject fluids. Those exemplifiedspecifically include a case where a purification processing of a proteinemploying column tips is conducted, a case where a purificationprocessing of a protein employing antibodies is conducted a purificationof a protein recombinant, and a cases where major proteins in a serumare removed.

In a processing where an antibody is employed to purify a protein, acolumn tip enclosed with an affinity gel such as Protein A and Protein Gas the packing is employed. After allowing a sample to be adsorbed ontothe packing a glycine-HCl buffer solution was employed for elution,whereupon achieving the abovementioned purified antibody desalting ortrace low molecular weight contaminant removal by means of a gelfiltration chromatography.

In the case of purification of a recombinant protein, anti GST antibodyor glutathione for purification of a GST fusion protein or histidine tagfusion protein, and a column tip enclosed with an affinity gel on whicha nickel ion is immobilized are employed. After adsorbing a sample,elution is conducted using a salt solution at a high concentration or asolution of a reduced form of glutathione and an imidazole solution, andthen the crude fusion protein solution is subjected to desalting orremoval of low molecular weight contaminants using a gel filtrationchromatography.

When major proteins in a serum are to be removed, a column tip enclosedwith an affinity gel on which specific antibodies (several types) forthe major proteins are immobilized is employed. The serum is bound tothe specific antibodies by suction into and discharge from anequilibrated column tip, and after the final discharge the serumcomponents free of the major proteins are recovered.

A third aspect of the invention is a column tip processing devicewherein the controlling part has a designating part which designates thecolumn tips and a processing using the column tips, a requirementgenerating part which generates a structural requirement relating to thedesignated column tips and a processing requirement relating to thedesignated processing, and an optimum parameter determining part whichdetermines, based on the generated the structural requirement andprocessing requirement, an optimum parameter with which the suction anddischarge mechanism and the moving means should be in accordance.

The designating part is used for example in such a manner that a useruses a mouse to click a switch or indication on a display to select eachone of a plurality of column tips and a plurality of processings, or auser uses a keyboard to enter a number. Based on the column tip and theprocessing designated by the designating part, the structuralrequirement and the processing requirement were specified, and thenbased on the specified structural requirement and the processingrequirement an optimum parameter is determined for example by means ofarithmetic calculation.

A fourth aspect of the invention is a column tip processing device,wherein the optimum parameter determining part determines an optimumparameter relating to suction or discharge, based on the generatedcorresponding structural requirement and processing requirement for eachof the column tips and processing contents, in order to reduce thedifference in time between the suction or discharge operation startingtime for the suction and discharge mechanism and the fluid movementstarting time for column tips and also the offset of suction ordischarge operation quantity and the suction or discharge quantity ofthe fluid for the column tip after achieving the operation quantity.

As used herein, the “difference in time” is due to the fact that theliquid is not coming into a column tip immediately after operating asuction and discharge mechanism, such as a cylinder. The term “offset”means a differential quantity remaining between the actual liquidsuction or discharge quantity and the operational quantity of thesuction and discharge mechanism even after a sufficient time period haselapsed after completion of the operation of the suction and dischargemechanism. Accordingly, the suction or discharge operation quantity is adesired suction or discharge quantity complemented with the offset fluidquantity. In conjunction with the suction or discharge flow rate, thedifference in time can be reduced by suctioning the fluid slowly whenthe resistance of the fluid in the column tip is high. Furthermore, thesuction or discharge flow rate is selected while taking the reactivityand the reaction time of the ligand in the packing in the column tipwith the subject substance to be captured.

The optimum parameter determining part may be in such a manner that theoptimum parameter is determined based on a reference table which assignsto each of said column tips and processing contents an optimum parameterwhich was obtained previously while taking the generated correspondingstructural requirement and the processing requirement into account.

Such a reference table is provided for each of the designated columntips and processing contents, and based on the structural requirementand processing requirement obtained from the reference table anotherreference table indicating an optimum parameter is provided and storedin a memory. Thus, in the present invention, instead of obtainingpreliminarily each optimum parameter of the suction or discharge as afunctional value of a multivalent function of each structuralrequirement and processing requirement with regard to the structure of acertain column tip by means of a numerical calculation, an optimumsuction or discharge parameter, when each structural requirement andprocessing requirement are prescribed preliminarily, is specified bymeans of experiments whereby deriving the optimum parameter for therequirements.

Being based on “the structural requirement and processing requirement”naturally includes being based on the nature of the packing encompassedin the structural requirement and the details of the reaction with asubstance contained in the subject fluid encompassed in the processingrequirement.

A fifth aspect of the invention is a column tip processing device,wherein the optimum parameter determining means determines, based on astandard structural requirement which sets predetermined one or twostandard column tips and at least a part of the plurality of the itemsof the structural requirement corresponding to the standard processingcontents at one or more standard values and a standard processingrequirement which sets at least a part of the plurality of the items ofthe processing requirement at one or more standard values, an optimumparameter corresponding to structural requirements and processingrequirements other than the standard structural requirements and thestandard processing requirement.

For predetermined one or more types of standard column tips, thestandard structural requirement and the standard processing requirementare measured or prescribed, and then based on the standard structuralrequirement and the standard processing requirement, a standard optimumparameter is obtained for example by an experiment. Based on this, theoptimum parameter value is changed depending on the comparison whetherit is higher or lower than the standard value for a certain column tip,or the optimum parameter is determined by calculation using aninterpolation.

Here, as “one or more types of standard column tips”, one or more typesof different types of column tips are provided, and for each type areference table is prepared, and for a certain column tip a column tipsimilar to that column tip is designated.

A sixth aspect of the invention is a column tip processing device,wherein the stage has a temperature raising and lowering vessel whichraises or lowers the temperature in response to an external signal, atleast one of the fluid housing part is housed in the temperature raisingand lowering vessel, and the control of the temperature of the subjectfluid is conducted on the moving means based on the processingrequirement.

Here, the temperature control is conducted by moving the nozzle to thetemperature raising and lowering vessel provided on the stage.

A seventh aspect of the invention is a column tip processing device,wherein on the stage one or more said column tips, one filter tip havinga fitting opening to be fitted to a nozzle, or one dispensing tip, and adetaching part for detaching the column tip, filter tip or dispensingtip fitted to the nozzle are provided, and the controlling partconducts, on the suction and discharge mechanism and the moving means,the control of the fitting and detachment of the column tip, filter tipor dispensing tip based on the structural requirement and the processingrequirement.

As used herein, “two or more types of column tips” may be in the caseswhere the packing is different, where the filter is different, where theshape of the tip-shaped vessel is different, and where even if thepacking is the same the ratio to the column tip capacity is different.Those which can be exemplified are a column tip for affinitychromatography in which a salt concentration is used to allow a proteinto be adsorbed and a column tip enclosed with an agarose gel fordesalting. The “filter tip” is one in which a filter is provided in atip-shaped vessel in such a manner that it serves as a partition betweenthe fitting opening and the hollow end. The “dispensing tip” is atip-shaped vessel which allows a magnetic field to be applied to theinside from the outside. When using a dispensing tip, it is required toprovide the nozzle head with a magnetic force means capable of applyinga magnetic field to the inside of the dispensing tip.

An eighth aspect of the invention is a column tip processing device,wherein at least a part of the fluid housing part group is provided witha piercable thin film covering the opening of the fluid housing part,the nozzle head is provided with a piercing pin capable of piercing thethin film, and the control of the thin film piercing is conducted on themoving means based on the processing requirement.

A ninth aspect of the invention is a column tip processing device,wherein the nozzle head is provided with a fall off preventing partwhich prevents the fall off from the nozzle by engaging with thetip-shaped vessel of the column tip fitted to the nozzle, and thecontrol of the fall off prevention and a cancellation thereof by thefall off preventing part is conducted on the moving means based on thestructural requirement and the processing requirement.

Here, since the fall off preventing part engages with the tip-shapedvessel of the column tip, it can be applied also to a dispensing tipenclosed with no packing or to a filter nozzle when using an identicaltip-shaped vessel. Such a fall off prevention and a cancellation thereofby the fall off preventing part is accomplished by moving control by themoving means.

A tenth aspect of the invention is a column tip processing methodhaving: based on a structural requirement of one or more types of columntips each having a tip-shaped vessel having a fitting opening conductingsuction or discharge of a gas by a suction and discharge mechanism and ahollow end through which a fluid can flow in or out in response to thesuction or discharge of the gas and a packing enclosed in the tip-shapedvessel, and a processing requirement relating to the processing contentsinvolving one or more types of subject fluids subjected to the suctionor discharge of the column tips, a fitting step for fitting a column tipto the nozzle at the fitting opening by moving the nozzle relativelybetween it and the housing parts of the column tip housed therein; acontacting step for inserting the hollow end into the fluid housing partby a relative movement between one or more fluid housing part housingthe subject fluid and the nozzle whereby suctioning and discharging thesubject fluid according to quantities, pressure, flow rate, the numberof cycles, time or position of the suction or discharge by the nozzledetermined based on the requirement, whereby bringing the packing intocontact with the subject fluid; and a discharging step for dischargingthe subject fluid from the column tip into one or more the fluid housingparts.

When in the column tip, a liquid for maintaining the packing activationis enclosed in a breakable manner together with the packing in such amanner that each of the fitting opening and the hollow end is closed ina removable state by the upper lid and the lower cap, then the capremoving step is provided after the fitting step.

An eleventh aspect of the invention is a column tip processing method,wherein the packing is enclosed in the tip-shaped vessel using at leastone filter provided in a manner to partition the tip-shaped vessel, thestructural requirement includes a plurality of items relating to thestructure of the member or the tip-shaped vessel fitted to the nozzle,the structure of the filter or the morphology, type and nature of thepacking enclosed, and the processing requirement includes a plurality ofitems relating to the processing contents including each housingposition, type, nature or quantity of one or more types of the subjectfluids subjected to the suction or discharge of the column tips.

A twelfth aspect of the invention is a column tip processing method,further having a designating step for designating column tips to befitted and processing contents to be processed, and a generating stepfor generating a structural requirement corresponding to the column tipsdesignated and generating a processing requirement corresponding to theprocessing contents designated, wherein the contacting step has anoptimum parameter determining step for determining an optimum parameterbased on the generated structural requirement and processingrequirement, and a contact executing step for moving the nozzle whileexecuting suction or discharge.

A thirteenth aspect of the invention is a column tip processing method,wherein the optimum parameter determining step determines suction ordischarge parameters, based on the generated corresponding structuralrequirement and processing requirement for each of the column tips andprocessing contents, while considering the difference in time betweenthe suction or discharge operation starting time for the suction anddischarge mechanism and the fluid movement starting time for columntips, the offset of suction or discharge operation quantity and thesuction or discharge quantity of the fluid for the column tip afterachieving the operation quantity, and the details of the reaction of thepacking with substances contained in the subject fluid, in order tocorrect the difference between the targeted suction or dischargequantity at the completion of the suction or discharge and the suctionor discharge quantity of the fluid for the column tip after completionof the operation.

A fourteenth aspect of the invention is a column tip processing method,wherein the optimum parameter determining step determines, based on astandard structural requirement for which one or more standard valuesare set for at least a part of the plurality of the items of thestructural requirement generated corresponding to the predeterminedstandard column tips and the standard processing contents and a standardprocessing requirement for which one or more standard values are set forat least a part of the plurality of the items of the processingrequirement, an optimum parameter corresponding to structuralrequirements and processing requirements other than the standardstructural requirements and the standard processing requirement forthose other than the predetermined standard column tips and processingcontents.

A fifteenth aspect of the invention is a column tip processing methodaccording to Claim 10, comprising, after the discharge step, a step forwashing the packing enclosed in the column tip by allowing a washingfluid as a subject fluid to be suctioned into and discharged from thecolumn tip and an elution step for introducing an eluent into the columntip whereby eluting the processing subject fluid-carried biologicalsubstances which were adsorbed to, captured by and reacted with or boundto the packing.

A sixteenth aspect of the invention is a column tip processing method,wherein, in the contact step, a temperature raising and lowering stepfor raising and lowering the temperature of the subject fluid based onthe processing requirement is provided, and the raising and lowering ofthe temperature is conducted by means of a relative movement between thenozzle and the temperature raising and lowering vessel provided on thestage.

A seventeenth aspect of the invention is a column tip processing method,further including a detachment step for detaching the column tips fittedto the nozzle, wherein the detaching step is conducted by means of arelative movement between the detaching part provided on the stage andthe nozzle based on the structural requirement and processingrequirement.

When, upon the fitting step, the column tip is subjected to a step forpreventing the fall down from the nozzle for the purpose of preventingthe fall off from the nozzle for example by engaging a fall offpreventing member in the form of a clamp or a comb with the protrusionor the stage of the column tip, it is required, before the detachingstep, to pass through a step for cancelling the fall off prevention bybreaking the engagement. Breaking the engagement may be accomplished forexample by moving the nozzle.

An eighteenth aspect of the invention is a column tip processing method,wherein, after the detaching step, fitting of at least one said columntip of another type, one filter tip connectable to the nozzle, or adispensing tip housed on the stage is conducted, based on the structuralrequirement and processing requirement, by means of a relative movementbetween the nozzle and the housing part in which the column tip, filtertip or dispensing tip is housed.

A nineteenth aspect of the invention is an optimum parameter generatingprogram, for a column tip processing device including: a nozzle headhaving a single or multiple-channeled nozzle; a suction and dischargemechanism conducting suction or discharge of a gas via the nozzle; oneor more types of column tips each having a tip-shaped vessel having afitting opening to be fitted to the nozzle and a hollow end throughwhich a fluid can flow in or out in response to the suction or dischargeof the gas and a packing enclosed in the tip-shaped vessel; a stageprovided with a fluid housing part group into which the hollow end canbe inserted and which houses or can house various solutions; and movingmeans for moving the nozzle head relatively to the fluid housing partgroup, wherein the optimum parameter generating program incorporates oneor more column tips fitted to the nozzle and a designating data whichdesignates the processing conducted using the column tips, generates,based on the designating data, a structural requirement data relating tocorresponding column tip structure and a processing requirement datarelating to the processing contents involving one or more types ofsubject fluids subjected to the suction or discharge of the column tipsincluded in corresponding processing, and determines, based on therequirements generated, optimum parameter data prescribing quantities,pressure, flow rate, the number of cycles, time or position of thesuction or discharge by the nozzle for the suction and dischargemechanism and the moving means.

A twentieth aspect of the invention is an optimum parameter generatingprogram, wherein the packing is enclosed in the tip-shaped vessel usingat least one filter provided in a manner to partition the tip-shapedvessel, the structural requirement data include a plurality of itemsrelating to the structure of the member or the tip-shaped vessel to befitted to the nozzle, the structure of the filter or the morphology ornature of the packing enclosed, and the processing requirement datainclude a plurality of items relating to the processing contentsincluding each housing position, nature or quantity of one or more typesof the subject fluids subjected to the suction or discharge of thecolumn tips.

A twenty first aspect of the invention is an optimum parametergenerating program, wherein the optimum parameter decision determines anoptimum parameter data relating to suction or discharge, based on thegenerated corresponding structural requirement data and processingrequirement data for each of the column tips and processing contents,while considering the difference in time between the suction ordischarge operation starting time for the suction and dischargemechanism and the fluid movement starting time for column tips, theoffset of suction or discharge operation quantity and the suction ordischarge quantity of the fluid for the column tip after achieving theoperation quantity, and the details of the reaction of the packing withsubstances contained in the subject fluid, while considering the suctionor discharge parameter and the difference in time between the suction ordischarge operation starting time and the fluid movement starting timefor column tips, the offset of suction or discharge operation quantityand the suction or discharge quantity of the fluid for the column tipafter achieving the operation quantity, and the details of the reactionof the packing with substances contained in the subject fluid.

A twenty second aspect of the invention is a column tip processingdevice including: a nozzle head having a single or multiple-channelednozzle; a suction and discharge mechanism conducting suction ordischarge of a gas via the nozzle; one or more types of column tips eachformed from a tip-shaped vessel having a fitting opening fitted orcapable of being fitted to the nozzle and a hollow end through which afluid can flow in or out in response to the suction or discharge of thegas and each accompanied with a packing enclosed in the tip-shapedvessel, or one or more types of filter tips each formed from thetip-shaped vessel and having a filter therein provided in a manner topartition between the fitting opening and the hollow end; a stageprovided with a plurality of fluid housing parts into which the hollowend can be inserted and which houses or can house various solutions anda tip housing part group capable of housing the column tips or filtertips; moving means for moving the nozzle head relatively to the fluidhousing part group; and a detaching part for detaching the column tip orfilter tip fitted to the nozzle, wherein the control of the fitting anddetachment of the column tip or filter tip is conducted on the suctionand discharge mechanism and the moving means based on the processingrequirement relating to the column tip and filter tip.

A twenty third aspect of the invention is a column tip processingdevice, including one or more types of dispensing tips formed with thetip-shaped vessel and capable of adsorbing magnetic particles onto aninner wall by means of a magnetic field given internally and magneticmeans provided in the nozzle head and capable of allowing a magneticfield to be applied to or removed from the inside of the dispensing tipfitted, wherein the dispensing tips can be housed in the tip housingpart group provided on the stage, the detaching part can detach thedispensing tips, and the control of the fitting and detachment of thedispensing tip, by the controlling part, is conducted on the suction anddischarge mechanism and the moving means based on the processingrequirement relating to the dispensing tip.

A twenty fourth aspect of the invention is a column tip processingdevice, wherein said one or more types of the column tips comprise gelfiltration column tips or affinity column tips.

As used herein, a “gel filtration column tip” is, among column tips, atip whose packing enclosed is a gel for a gel filtration chromatographysuch as Sephadex or Sephacryl, and serves for a processing in such amanner that a subject fluid is introduced from the fitting opening ofthe column tip and the subject fluid is allowed to pass through thepacking by gravity or under pressure whereby discharging the liquid tothe outside of the column tip. Only one filter is provided under thepacking. The “affinity column tip” means a tip enclosed with a gel foran affinity chromatography to which a substance reacting specificallywith an intended biological substance such as an antibody, antigen,nickel, glutathione and the like is bound.

A twenty fifth aspect of the invention is a column tip processingdevice, wherein the nozzle head is provided with a fall off preventingpart which prevents the fall off from the nozzle by engaging with thetip-shaped vessel of the column tip fitted to the nozzle, the control ofthe fall off prevention and a cancellation thereof by the fall offpreventing part is conducted on the moving means based on the processingrequirement.

A twenty sixth aspect of the invention is a column tip processingmethod, in which a stage houses one or more types of column tips eachhaving a tip-shaped vessel having a fitting opening fitted to a singleor multiple-channeled nozzle, conducting suction or discharge of a gasby a suction and discharge mechanism and a hollow end through which afluid can flow in or out in response to the suction or discharge of thegas and a packing enclosed in the tip-shaped vessel, or one or moretypes of filter tips each having the tip-shaped vessel and a filterprovided in a manner to partition between the fitting opening and thehollow end, and, which comprises, based on a processing requirementrelating to the processing contents involving one or more types ofsubject fluids subjected to the suction or discharge of the column tipsor filter tips, a fitting step for fitting a column tip or filter tip tothe nozzle at the fitting opening by moving the nozzle relativelybetween it and the housing parts of the column tip or filter tip housed;a suctioning and discharging step for inserting the hollow end into thefluid housing part by a relative movement between one or more fluidhousing part housing the subject fluid and the nozzle whereby suctioningand discharging the subject fluid; a detaching step for detaching columntips or filter tips fitted to the nozzle; and, a re-fitting step forconducting fitting of at least one said column tip of another type orone filter tip housed on the stage to the nozzle is conducted by meansof a relative movement between the nozzle and the housing part in whichthe column tip or filter tip is housed.

A twenty seventh aspect of the invention is a column tip processingmethod which houses, on the stage, one or more types of dispensing tipseach formed from a tip-shaped vessel and capable of adsorbing magneticparticles onto inner wall by means of a magnetic field given internally,

wherein the processing requirement relates to the processing contentsinvolving one or more types of subject fluids subjected to the suctionor discharge of a dispensing tip, the fitting step comprises a step forfitting the dispensing tip to the nozzle, the suction or discharge stepcomprises a step for suctioning and discharging the subject fluid whileapplying a magnetic force into the dispensing tip, wherein the detachingstep comprises a step for detaching the dispensing tip fitted to thenozzle, and the re-fitting step conducts fitting of the dispensing tipto the nozzle is conducted by means of a relative movement between thenozzle and the housing part in which the dispensing tip is housed.

A twenty eighth aspect of the invention is a column tip processingmethod, wherein said one or more types of the column tips comprise gelfiltration column tips or affinity column tips.

A twenty ninth aspect of the invention is a column tip processing deviceincluding: a nozzle head having a single or multiple-channeled nozzle; asuction and discharge mechanism conducting suction or discharge of a gasvia the nozzle; one or more types of column tips each formed from atip-shaped vessel having a fitting opening fitted or capable of beingfitted to the nozzle and a hollow end through which a fluid can flow inor out in response to the suction or discharge of the gas and eachaccompanied with a packing enclosed in the tip-shaped vessel; a stageprovided with a plurality of fluid housing part group into which thehollow end can be inserted and which houses or can house varioussolutions; and moving means for moving the nozzle head relatively to thefluid housing part group, wherein, in the fluid housing part group, abuffer solution having a plurality of different concentrations arrangedat a certain concentration difference from each other along with acertain concentration gradient with regard to a certain salt is housed.

The “arranged” form may for example be a matrix form or a linear form.

A thirtieth aspect of the invention is a column tip processing device,wherein on the stage, the column tips, and dispensing tip are housed,and a detaching part for detaching the fitted dispensing tip or columntip from the nozzle are provided.

A thirty first aspect of the invention is a column tip processingmethod, for one or more types of column tips each having a tip-shapedvessel having a fitting opening, fitted to a single ormultiple-channeled nozzle, conducting suction or discharge of a gas by asuction and discharge mechanism and a hollow end through which a fluidcan flow in or out in response to the suction or discharge of the gasand a packing enclosed in the tip-shaped vessel, which includes: anadsorption step for allowing an intended biological substance to beadsorbed onto the packing by means of suction and discharge of a subjectfluid containing the intended biological substance; and a contactingstep for housing, as a subject fluid subjected to the suction ordischarge of said two or more column tips, a buffer solution having aplurality of different concentrations arranged at a certainconcentration difference from each other along with a certainconcentration gradient with regard to a certain salt, and inserting oneor more column tips into one or more said fluid housing part to effectsuction or discharge, whereby bringing the packing into contact with thebuffer solution.

A thirty second aspect of the invention is a column tip processingmethod, wherein generation and housing of the buffer solution isconducted using a dispensing tip fitted to the nozzle.

ADVANTAGE OF THE INVENTION

According to the first, tenth, or nineteenth aspect of the presentinvention, for one or more types of column tips, based on a structuralrequirement relating to the structure of the column and a processingrequirement relating to the processing contents involving one or moretypes of subject fluids subjected to the suction or discharge,quantities, pressure, flow rate, and the like of the suction ordischarge by the nozzle are determined. As a result, a highly efficientprocessing can be conducted by bringing the subject fluid into contactwith the packing in a manner of suction or discharge which is optimumfor the column tip having a complicated structure containing varioustypes of packing. In addition, by conducting the suction or dischargewith a small amount of the liquid including only the subject fluidwithout using a mobile medium including a large quantity of a buffersolution and without diluting an intended biological substance, a highefficiency with a small-scaled device can be accomplished.

It is also possible, based on the structural requirement and processingrequirement, a continuous automatic processing including fitting ofcolumn tips to the nozzle, selection of optimum column tips, selectionof a necessary subject fluid, transportation to the position of housing,and optimum suction or discharge can be accomplished.

According to the second, eleventh, or twentieth aspect of the presentinvention, by taking the structure of the tip-shaped vessel, thestructure of the filter or the shape or the nature of the packing intoaccount as structural requirements with regard to the column tips, andalso by taking the nature and the quantity of the subject fluid, thefactors serving as a resistance against the suction or discharge of thefluid are contemplated, whereby controlling optimum pressure, flow rate,cycles or time period of the suction or discharge.

According to the third, twelfth, or nineteenth aspect of the presentinvention, a structural requirement relating to the designated columntips and a processing requirement relating to the designated processingare generated, and based on such structural requirement and processingrequirement an optimum parameter is determined. Accordingly, only adesignation by a user of the column tips and the processing enablesvarious structural requirements and the various requirements for theprocessing to be taken into account automatically, whereby allowing theoptimum parameters to be readily obtained.

According to the fourth, thirteenth, or twenty first aspect of thepresent invention, since an optimum parameter is determined based on thedifference in time between the operation designating time and the fluidmovement starting time, the suction or discharge operation quantity, andthe offset of the suction or discharge quantity of the fluid for thecolumn tip after achieving the operation quantity, the optimum parametercan quickly and readily be obtained, and various cases can readily behandled just by exchanging the reference table.

According to the fifth or fourteenth aspect of the present invention, byprescribing a standard structural requirement and a standard processingrequirement corresponding to the predetermined standard column tip andthe standard processing contents, an optimum parameter corresponding toother structural requirements and processing requirements can readily beobtained. Because of a simple construction applicable to various columntips and processings, a wide range of use is possible.

According to the sixth or sixteenth aspect of the present invention, byconducting the control of the temperature of the subject fluid to be incontact with the packing, by conducting the control of the temperatureof the subject fluid to be in contact with the packing by means of themovement to the temperature raising and lowering vessel based on theprocessing requirement, the temperature control and the suction ordischarge can be conducted independently in parallel, whereby allowingthe suction or discharge to be conducted at an optimum temperature. Inaddition, since the temperature is controlled by moving, the control canreadily be conducted.

According to the seventh or seventeenth aspect of the present invention,by providing a detaching part for a column tip once fitted to a nozzle,a plurality of s of column tips, filter tips or dispensing tips cansequentially be exchanged by a single device, or can simultaneously beemployed in combination, whereby allowing diverse processings to beconducted continuously and automatically.

According to the eighth aspect of the present invention, by providingnecessary reagents preliminarily in a pre-packed vessel, the necessaryreagents can be handled readily, rapidly and efficiently, only by movingthe nozzle, in a best and highly reliable state when necessary.

According to the ninth aspect of the present invention, by engaging witha tip-shaped vessel fitted to a nozzle, a column tip is prevented fromfalling off from the nozzle of the column tip. Therefore, it becomespossible to apply a high pressure, against the presence of the packingor the filter, to the column tip upon discharge, whereby enablingapplication to various column tips and filter tips. The cancellation ofthe fall off prevention can readily and automatically be conducted bymovement of the moving means. As a result, the column tip or the likecan be detached, and various column filters or other tips can beemployed as being exchanged or in combination.

According to the fifteenth aspect of the present invention, by a stepfor washing the packing and eluting the biological substance onceadsorbed onto the packing, contaminants which were not adsorbed onto thepacking can surely be removed by repeating suction and discharge, andthe biological substance adsorbed on the packing can only be recoveredsurely and efficiently.

According to the twenty second or twenty sixth aspect of the presentinvention, since the column tip or the filter tip fitted to the nozzlecan be detached and then other types of tips can further be fitted, aplurality of types of column tips or filter tips can sequentially beexchanged, or can simultaneously be employed in combination in a singledevice, whereby allowing diverse processings to be conductedcontinuously and automatically.

According to the twenty third or twenty fifth aspect of the presentinvention, since the dispensing tip fitted can further be detached andthen other types of tips can further be fitted, or other types of thetips are detached and the dispensing tip can be fitted to conduct aprocessing. Accordingly, a plurality of types of tips includingdispensing tips can sequentially be exchanged, or can simultaneously beemployed in combination in a single device, whereby allowing diverseprocessings, including absorption using a magnetic field, to beconducted continuously and automatically.

According to the twenty fourth or twenty seventh aspect of the presentinvention, by using the above type of the column tips as a column tip,for example by using a dispensing tip and a gel filtration column tip, amagnetic particle-based fusion protein purification and a pretreatmentof gel filtration column tip-based protein analysis can continuously beperformed. In addition, by combining the affinity column tip and the gelfiltration column tip, the affinity column tip is used for removing themajor proteins, whereby enabling the extraction of the useful proteinscontinuously. By combining the affinity column tip, the gel filtrationcolumn tip and the filter tip, a pretreatment of a protein analysissample can continuously be conducted.

According to the twenty ninth or thirty first aspect of the presentinvention, instead of a conventional separation based on the specificityand the nature of a protein resulting from a difference in theconcentration along with the time, by housing a set of a plurality ofbuffer solutions having differences in concentration from each otheralong with a gradient of the concentration preliminarily in fluidhousing parts and then moving the column tips between the fluid housingparts, or by inserting a plurality of the column tips at once into thefluid housing parts, or by combining these procedures, the concentrationat which the intended biological substance is eluted surely at a highaccuracy with the desired concentration difference.

According to the thirtieth or thirty second aspect of the presentinvention, since a set of a plurality of buffer solutions havingdifferences in concentration from each other along with a gradient ofthe concentration can be subjected to fitting of dispensing tips to anozzle of an identical device, the processing can readily andcontinuously be conducted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a column tip processing device accordingto a first embodiment of the invention.

FIG. 2 is a side view of a nozzle head of the column tip processingdevice according to the first embodiment of the invention.

FIG. 3 is a planar view of the column tip processing device according tothe first embodiment of the invention.

FIG. 4 is a sectional view of a column tip before fitting to the nozzleaccording to the first embodiment of the invention.

FIG. 5 shows suction and discharge of a column tip according to thefirst embodiment of the invention.

FIG. 6 shows suction and discharge of a column tip according to a secondembodiment of the invention.

FIG. 7 shows suction and discharge of a column tip according to a thirdembodiment of the invention.

FIG. 8 shows other column tips of the invention.

FIG. 9 is a schematic view of an optimum parameter determining part of acontrolling part according to the second embodiment of the invention.

FIG. 10 is a schematic view of an optimum parameter determining part ofa controlling part according to the second embodiment of the invention.

FIG. 11 is a flow chart indicating a column tip processing according toa fourth embodiment of the invention.

FIG. 12 is a flow chart of a column tip processing according to a fifthembodiment of the invention.

FIG. 13 is a flow chart of a column tip processing according to a sixthembodiment of the invention.

FIG. 14 is a flow chart of a column tip processing according to aseventh embodiment of the invention.

FIG. 15 shows a view of a fluid housing part group according to aneighth embodiment of the invention as well as the concentrations of thefluids to be housed.

BEST MODE FOR CARRYING THE INVENTION

A column tip processing device 10 according to a first embodiment of theinvention is described with reference to FIGS. 1 to 3.

The column tip processing device 10 includes a nozzle head 15 having asuction and discharge mechanism 16 for conducting the suction anddischarge of a gas and a multiple-channeled (in this case, 12-channeled)nozzle 14 to which column tips 12 are fitted as described below andwhich is arranged linearly along with Y axis for allowing the gas to besuctioned into or discharged from these column tips 12, a stage 13having a housing part group 20 having, in the form of a row, variousfluid housing part which houses various samples or reagent-containingsolutions to be suctioned into the column tips 12 or discharged from thecolumn tips 12 or a tip housing part 61 which houses various column tipsincluding the column tips 12, a moving mechanism 97 as a moving meansfor moving the nozzle head 15 and the housing part group 20 relativelyin a horizontal direction (in this case, in the direction of X axis) andin a vertical direction (in the direction of Z axis), an operation panel24 and a lid-fitted IC card insertion port 30 for designating the columntips and the processing contents and for entering various data, adetachment plate 42, provided on the stage 13, as a detaching part fordetaching the column tips 12 from the nozzle 14, and a case 22 in whichthe column tips and respective components are housed in a mannerallowing them to be operated externally. Here, the X axis, Y axis and Zaxis together form a three dimensional rectangular coordinate system.The operation panel 24 and the lid-fitted IC card insertion port 30together with an information processing part (not shown) constitute acontrolling part.

On the operation panel 24, a liquid crystal display 28 and an inputkeyboard 26 are provided. On the nozzle head 15, 12 magnets, as amagnetic force means for applying a magnetic force to the inside of adispensing tip (110, see FIG. 8) when this dispensing tip is fitted anozzle 14, are provided as being arranged on a rod-shaped memberextending longitudinally (in the direction of the Y axis in the figure)at an interval of the arrangement of the nozzle 14 while capable ofbeing in contact with and released from the dispensing tip in aback-and-forth direction (in the direction of the X axis in the figure).

A fall off preventing parts 33 to 38, 40, 44 and 46 serve to prevent thefall off of the column tips 12, that are fitted as being engaged withthe nozzle 14, from the nozzle 14, or serve to cancel such a fall offprevention, and are provided in such a manner that they move in concertwith the nozzle head 15 movable in the direction of the Z axis. Thisfall off preventing part is provided since a high pressure should beapplied by the suction and discharge mechanism to the column tips 12because of the structure inside of the column tips 12 serving as aresistance to a fluid, such as the packing, filters and the like. On thestage 13, two wall plates 21 extending in the direction of the X axis assandwiching the housing part group 20 are provided. A screen 29 is tohide a P axis motor 49 and the like provided in the back.

FIG. 2 shows a side view of the major part including the nozzle head 15placed in the column tip processing device 10.

The 12 individual nozzles 14 of this nozzle head 15 are supported by anozzle supporting member 54 as being arranged along with thelongitudinal direction thereof.

Above each individual nozzle 14, 12 cylinders 47 in communication withthe nozzles 14 are provided, and inside of each cylinder 47 a plunger 43is provided pivotally each in the direction of the axis, and the top 51of the upper end of the plunger 43 is supported as being fixed on thedriving plate 45 in a state that the top 51 is protruded over thisdriving plate 45 which is driven up and down by the P axis motor 49 forthe suction and discharge which is provided as being supported by a Zaxis moving body 27 provided in the nozzle head 15. On this drivingplate 45, 12 hexangular prism-shaped hexangular posts 50 are attached inthe position at a certain distance in the direction of the X axis fromthe plunger row where the plungers 43 are arranged in the direction ofthe Y axis while being arranged in the direction of the Y axis at aninterval of the arrangement identical to that of the plungers 43 andalso being allowed to extend downward. At the lower end of thehexangular post, a puncturing needle 48 for puncturing a thin film forexample of a prepack is protruded downwardly.

The driving plate 45 is fixed to a nut part 17 which is engaged with aball screw 19 driven to rotate via a coupler 49 a by the P axis motor 49for suction and discharge provided as being supported by the Z axismoving body 27 of the nozzle head 15 supported movably in the directionof the Z axis relatively to a fixed case 22 of the column tip processingdevice 10, and also which moves up and down in response to the rotationof this ball screw 19. This ball screw 19 is supported coaxially to theZ axis moving body 27 by means for example of a bearing.

This Z axis moving body 27 is provided movably in the direction of the Zaxis as being guided by a Z axis moving body supporting platform 81provided as being fixed to the case 22. On the top of this Z axis movingbody supporting platform 81, a Z axis motor 77 is provided, and servesto move this Z axis moving body 27 in conjunction via a coupler 77 awith the ball screw which engages with the nut part provided as beingfixed on the Z axis moving body 27. The stage 13 is formed above an Xaxis moving body provided movably in the direction of the X axis asbeing set as described above. On a base 92 of the case 22, a linearmotion rail 93 is laid in the direction of the X axis, and beneath the Xaxis moving body 94, linear motion guides 95 a and 95 b on which aplurality of balls are supported as being capable of rotating andcirculating are provided, and are capable of moving smoothly as beingguided by the linear motion rail 93. Here, the Z axis moving body 27,the Z axis moving body supporting platform 81, the X axis moving body 94and the rail 93 and the like correspond to the moving means 97.

On this driving plate 45, a light shielding plate (not shown) isprovided as being extending vertically. When the driving plate 45reaches the upper limit of the plunger 43, then blocks the light from alight emitting element provided as being fixed to the case 22, wherebypreventing the arrival at the light receiving element.

Beneath each cylinder 47 described above, a horizontal plate 54 a havingpores (not shown), whose size allows the passage of the nozzle but doesnot allow the passage of the column tip 12 or the dispensing tip 110(see FIG. 8), provided under the nozzle supporting member 54 along withthe longitudinal direction of this nozzle supporting member 54 isprovided. On each of the both ends of this horizontal plate 54 a, atubular post 52 is each provided which supports the horizontal plate fordetaching the dispensing tips (not column tips). The 12 cylinders 47 arefixed at their upper ends by a cylinder supporting member 52 a whilebeing supported to extend downward therefrom, and the post 52 penetratesthis cylinder supporting member 52 a to protrude upward therefrom, andthe upper end of the post 52 is supported by a post supporting tube 53as being fixed. The 12 plungers 43 penetrate the post supporting tube53, and are slidable independently. The cylinder supporting member 52 ais provided in such a manner that it moves in conjunction with thenozzle head 15.

The driving plate 45 functions, when conducting the suction anddischarge of a gas or when puncturing a thin film of such as a prepackusing the puncturing needle 48, by moving back and forth on the plunger43 between the upper limit position and the upper end of the postsupporting tube 53, while the driving plate 45 can go down further fromthe upper end of the post supporting tube 53 to reach the lower limitposition. In such a case, the driving plate 45 goes down together withthe post supporting tube 53 whereby pushing the post 52 and allowing thehorizontal plate to go down, whereby allowing the dispensing tip oncefitted (not used for the column tips) to be drawn down apart from thenozzle 14. The detachment of the column tip 12 from the nozzle 14 isdiscussed below.

Based on FIGS. 1 and 2, the mechanism possessed by the fall offpreventing part which prevents the column tips 12 from falling off fromthe nozzle 14 is described.

In this fall off preventing part, a fall off preventing member 35 having12 approximately semicircular notches (not shown) arranged in thepositions and at an interval of the arrangement identical to that of thecolumn tips 12 in the direction of the Y axis is supported rotatably ona horizontal rod 33. The size of each notch is slightly larger than thediameter of the tube having a large diameter 64 of the column tip 12 andsmaller than the outer diameter including the thickness of a protrudingstreak 74 of this column tip, and in this manner the column tip 12 canbe supported at the position of this protruding streak 74. Thishorizontal rod 33 is provided in such a manner that it moves inconjunction with the nozzle head 15, and this horizontal rod 33 isprovided in such a manner that it can move up and down along with theguide rail provided as being fixed in the case 22. This horizontal rod33 is kept in a rod supporting member 34 in such a manner that itpenetrates a oval hole 34 which allows the horizontal rod 33 to rotateand to move over a slight distance in the direction of the X axis. Inthis rod supporting member 34, two guide parts 36 penetrate theanchoring block 41 formed from a permanent magnet and extend thereover,and on the top of it an upper end part 44 formed from a magnetic body isprovided. On the upper end part 44, a knob 44 a for releasing the closecontact with the anchoring block 41 due to the magnetic force isprovided. In the state where the fall off preventing member 35 isengaged with the column tip 12 whereby preventing the fall of the columntip, the upper end part 44 is in a close contact with the anchoringblock 41 due to the magnetic force and gravity, as shown in FIG. 2.

In the fall off preventing member 35, in the middle between the end incontact with the column tip 12 and the horizontal rod 33, the fall offpreventing member 35 is supported at its axis rotatably on an invertedL-shaped member whose side view is in the shape of an inverted L via apin 31. This inverted L-shaped member 37 is provided on each of the bothends of an anchoring block supporting member 38 which support theanchoring block 41 which is formed as extending in the direction of theY axis similarly to the nozzle supporting member 54. Above thisanchoring block supporting member 38, 2 upwardly extending guide parts40 are formed from a permanent magnet, penetrate an anchoring horizontalrod 39 fixed on the Z axis moving body 27 of the nozzle head and extendthereover, and on the top of it an upper end part 46 formed from amagnetic body is provided. In addition, on this upper end part 46, aknob 46 a for releasing the close contact with the anchoring horizontalrod 39 due to the magnetic force is provided. In the state where thefall off preventing member 35 is engaged with the column tip 12 wherebypreventing the fall of the column tip, the upper end part 46 is in aclose contact with the anchoring horizontal rod 39 due to the magneticforce and gravity, as shown in FIG. 2.

In order to release the state of the engaging of the fall off preventingmember 35 with the column tip 12, thus, the state of the fall offprevention, the X axis moving body 94 is moved in the direction of the Xaxis, whereby moving the nozzle head 15, by means of the movingmechanism, relatively in the direction of the X axis to come above awall part 23 for release, among the wall plate 21, which is mounted asbeing slightly higher than others in the direction of the Z axis, andthereafter using the moving mechanism the nozzle head 15 is moveddownward in the direction of the Z axis. As a result, the horizontal rod33 is brought into contact with the wall part 23 for release, and upon afurther downward movement, this horizontal rod 33 receives an upwardforce whereby rotating around the pin 31 as an axial fulcrum, resultingin release of the notch of the fall off preventing member 35 from theprotruding streak 74. Upon this, since the parts other than thehorizontal rod 33 and the part attaching thereto are going to movefurther downward, the upper end part 44, which are attached via the rodsupporting member 34 and a guide member 36 to this horizontal rod 33 isreleased from the anchoring block 41. At the same time, the upper endpart 46, which are attached via inverted L-shaped member 37 and a guidemember 40 to this horizontal rod 33 is released from the anchoringhorizontal rod 39.

On the other hand, in order to validate the fall off prevention, the Zaxis moving body 27 of the nozzle head 15 is raised in the direction ofthe Z axis, whereby first raising the parts other than the horizontalrod 33 and the part attaching thereto, and the upper end part 44 isbrought into a close contact with the anchoring block 41 due to it's ownweight and a magnetic force, and the upper end part 46 is brought into aclose contact with the anchoring horizontal rod 39 due to it's ownweight and a magnetic force, and simultaneously the horizontal rod 33 isreleased from the wall part 23 for release, resulting in engagement ofthe fall off preventing member 35 with the protruding streak 74 of thecolumn tip 12, whereby accomplishing the fall off prevention.

FIG. 3 shows the stage 13 of the column tip processing device 10. Onthis stage 13, a reagent rack 55, as the housing part group 20, having12 groups of reagent prepack cartridges 58 including a plurality offluid housing parts in which a certain reagent solution haspreliminarily be housed and which has been covered with thin films and atip rack 18 having a 4-row 12 tip housing part 61 capable of housing thecolumn tips 12 or the dispensing tips are provided.

In the reagent prepack cartridge 58, a plurality of fluid housing parts(in this case, 10) 57 and heat block parts 56 for heating, and on eachof these heat block parts 56, a fluid housing part for the reagent and asample tube are provided. On the both sides of each reagent prepackcartridge 58, a septum 25 having a certain height is provided.

Among the tip racks 18, lower 2 rows house sample tubes 60. Above thetip housing parts for the upper 1 row, a detachment plate 42 as thedetaching part is provided. This detachment plate 42 has 12 holes 59,including holes 59 a each having a size enabling the passage of thenozzle 14 but not enabling the passage of the column tip 12 ordispensing tip and holes 59 b each having a size enabling the passage ofthe tube 66 having a small diameter of this column tip 12 but notenabling the passage of other parts, as being arranged on the detachmentplate 42 at an interval of the arrangement of the nozzle 14.

With reference to FIG. 4, the column tip 12 according to the firstembodiment is described.

The sectional view of this column tip 12 shown in FIG. 4 is in the statebefore fitting to the nozzle 14 of the column tip processing device 10.

This column tip 12 has a tip-shaped vessel 62 in which an opening 71 forfitting to be fitted to a nozzle 14 via the nozzle 14 or a member fittedto this nozzle 14 is provided at the upper end and a hollow end 69through which a fluid can flow in and out is provided at the lower end.This tip-shaped vessel 62 has a tube having a large diameter 64 providedwith the fitting opening 71 and enclosed with a packing 68, anapproximately cylindrical tube having a small diameter 66 formed asbeing narrower than this tube having a large diameter 64 and having thehollow end 69 at its end, and a step-like transfer part 73 formedbetween the tube having a large diameter 64 and the tube having a smalldiameter 66. The tube having a large diameter 64 has, in its lower side,a packing housing part 64 a into which the packing 68 is enclosed, and,in its upper side, a retaining part 64 b which is formed as beingbroader than the packing housing part 64 a and which can retain a fluid,as well as a step part 63 formed between this packing housing part 64 aand the retaining part 64 b. In addition, in the upper part of the outersurface of the tube having a large diameter 64, a plurality ofprotruding streaks 74 formed in the direction of the axis are provided,and employed for the support by a fall off preventing member 35 asdiscussed above. The packing 68 (for example, several ten micrometers to100 micrometers) is enclosed as being sandwiched by a filter 65 providedin the step part 63 and a filter 67 provided in the transfer part 73.

At the bottom of the packing housing part 64 a, a plurality ofprotrusions 67 a protruding in the direction of radius relative to theaxial line are provided as surrounding the axial line, wherebypreventing the filter 67 from being in a close contact.

The packing 68 of this column tip 12 should be stored in contact with anactivation maintaining fluid before the processing in order to maintainits activation.

Accordingly, the storage is in a state where for preventing any fluidleakage of the opening 71 for fitting is sealed by a lid 70 while thehollow end 69 is sealed by a cap 72.

Upon fitting to the nozzle 14, the lid 70 is removed manually andfitting is established by putting into the nozzle 14, while the cap 72is removed by inserting the tube having a small diameter 66 of thecolumn tip 12 laterally into the holes 59 b of the detachment plate 42,followed by moving the nozzle head 15 upward.

FIG. 5 shows the state where into the column tip 12 a subject fluid 76containing an intended biological substance is suctioned via the sampletube 60.

In this case, in the valid column tip region sandwiched between thefilter 65 and the filter 67, the entire volume of the packing 68occupies only about 60% of the capacity of the valid column tip region.In such a case, the quantity of the subject fluid 76 as a subject forsuction and discharge is suctioned and discharged repetitively to anextent not exceeding the upper filter 65, whereby establishing thesuspension state of the packing 68 with the subject fluid 76 withoutintroducing air, resulting in an enhancement of the contact between thepacking 68 and the biological substance contained in the subject fluid76. Such a procedure is employed mainly when enclosing an affinity gel.An exemplary use is for elution of a purified antibody after allowing asample to be adsorbed onto the affinity gel.

FIG. 6 shows a case where the column tip 78 has a tip-shaped vessel 62similar to the column tip 12 having a valid column region sandwichedbetween the similar filter 65 and filter 67, in which the entireparticulate volume of the packing 80 is enclosed as being compressed ata rate of about 100% of the valid column region.

In such a case, suction and discharge can be conducted in such a mannerthat the suction and discharge fluid quantity exceeds the capacity ofthe valid column tip region, and such a procedure is employed in aliquid chromatography in which the fractionation is conducted based onthe discharge time difference or the number of suction and dischargecycles. In addition, in this case, a microscopic suspension state isconsidered to be established. For example, a gel filtrationchromatography is employed for desalting of the purified antibody aswell as removing contaminants.

FIG. 7 shows a spinning top-like or disc-like column tip 82. This columntip 82 has a retaining part 84 having a fitting opening 91 capable ofbeing fitted to the nozzle 14, a hollow disk-shaped packing enclosedpart 86 in which the packing 85 is enclosed as being compressed at arate of about 100% of the capacity of the valid column region of theentire particulate volume, and a narrow tube 88 having a hollow end 83capable of being inserted into the sample tube 60.

This column tip 82 can reduce the suction and discharge time by makingthe thickness of the packing 85 in the direction of the axis thinner andincreasing the porosity, in the case where the particle size of thepacking 85 is small and the pore sizes of the filters 89 and 87 are alsosmall.

FIG. 8 shows another column tip 96, filter tip 102 and dispensing tip106, which can be fitted to the nozzle 14.

The column tip 96 is the tip-shaped vessel 62 enclosed with a gel for agel filtration chromatography such as Sephadex or Sephacryl using asingle filter 100. The filter tip 102 has a membrane filter or aultrafiltration membrane enclosed as a filter 104. With pressurizingfrom the top or natural pressurizing, elution can occur. When using anagarose gel to fractionate proteins based on the size, it is effectivefor increasing the accuracy to have a longer valid column length toenclose the packing.

The controlling part is discussed here.

This controlling part controls the suction and discharge mechanism 16and the moving means (not shown) with regard to quantities, pressure,flow rate, the number of cycles, time or position of the suction anddischarge by the nozzle 14 based on a structural requirement relating tothe structure of the one or more types of column tips 12, 78, 82, and 96to be fitted to the nozzle 14 and a processing requirement relating tothe processing contents involving one or more types of subject fluidssubjected to the suction and discharge of the column tips. Thestructural requirement includes a plurality of items relating to thestructure of the tip-shaped vessel 62 of the column tip, the structureof the filters 65, 67 or the morphology, and nature of the packingenclosed, and the processing requirement includes a plurality of itemsrelating to the processing contents including each housing position,type, nature or quantity of one or more types of the subject fluidssubjected to the suction and discharge of the column tips.

The controlling part has an operation panel 24 and a lid-fitted IC cardinsertion port 30 as a designating part for designating the column tipsand the processing using this column tip, a requirement generating partwhich generates a structural requirement relating to the designatedcolumn tips and a processing requirement relating to the designatedprocessing, and an optimum parameter determining part which determines,based on the generated the structural requirement and processingrequirement, an optimum parameter with which the suction and dischargemechanism and the moving means should be in accordance. The requirementgenerating part and the optimum parameter determining part are providedin the information processing device. This information processing devicehas a CPU, memories, software stored in a memory for controlling, whichare not shown in the figures.

Based on FIG. 9, an example that an optimum parameter determining partdetermines the optimum parameter based on the structural requirement andprocessing requirement generated by the requirement generating part isdescribed specifically below.

To determine these optimum parameter based on the structural requirementand processing requirement generated by the requirement generating partof the controlling part, various factors serving as a resistance againstsuction and discharge of a fluid, such as a packing 80, supportingfilter 67, 65 and the like, all provided in the column tip 78, should betaken into account.

FIG. 9( a) is a schematic view near the valid column region of thecolumn tip 78. Based on FIG. 9( a), examples of the structuralrequirement and the processing requirement generated by the requirementgenerating part are shown below. Among the structural requirement, onerelating to the structure of the tip-shaped vessel 62 includes thecolumn's inner diameter D, which is 5.1 mm in this case, the validcolumn length L (excluding a filter part), which is 2.5 mm in this case,the dead volume v corresponding to the capacity of the tube having asmall diameter 66 and the transfer part 73, which are not involved inthe reaction which is 50μ liters. One relating to the structure of thefilters 67 and 65 includes the filter thickness t, which is 1.4 mm inthis case and the porosity a of the filter 67. While the pore size ofthe filters 67 and 65 in this case is 80 to 120 μm (POREX, Hydrophobic),a particle smaller than the pore size can be retained due to itssubstantial thickness. One relating to the morphology of the packing 80in this case includes the particulate form, its mean particle size d,which is 90 μm in this case, while one relating to the type and thenature includes, for example, the type, NI Sepharose (GE HealthcareBioscience, NI Sepharose 6 Fast Flow). The processing requirementincludes the type of the subject fluid which is a suspension ofHis-Tag-GFP as a biological substance, the concentration which is 600μgram/600μ liters, and the quantity which is 650 μL. The quantity of thesubject fluid is 600μ liters. As processing contents, those included arethe case corresponding the adsorption of the subject fluid in Step S2shown in FIG. 10, where the biological substance described below isbrought into contact with the packing 80 to effect a binding reactionmovement.

The optimum parameter determining part determines the optimum parametersbased on the structural requirement and the processing requirement asdetailed above.

For determining the optimum parameters, it is a concern that a fluidresistance of the column tip 78 occurres for the respective factors. Thefluid resistance of the column tip 78 is mostly the sum of theresistance by the filter and the resistance by the packing. Theresistance by the packing is inversely proportional to the square of theratio to the standard inner diameter D, proportional to the standardvalid column length L, and proportional to the viscosity ν of thesubject fluid. On the other hand, the resistance by the filter isinversely proportional to the square of the ratio to the standard innerdiameter D, proportional to the ratio to the standard filter thickness,proportional to the ratio to the standard porosity, and proportional tothe viscosity ν of the subject fluid. In addition, there is a dependencealso on the processing contents of the processing requirement.

On the other hand, there is a time lag between the cylinder operation(solid line) and the fluid response (dotted line) as shown in FIG. 9( b)as a result of taking the fluid resistance into account. Such adifference is considered to be due mainly to the fluid resistance, inview of the understanding that there will be no time lag when suctioningand discharging an ideal fluid against zero resistance.

Thus, between the cylinder operation and the fluid response, there is atime lag of a “wasted time” corresponding to the difference in the timebetween the suction and discharge operation starting time for thesuction and discharge mechanism and the fluid movement starting time forcolumn tips, and the fluid does not enter the column tip immediatelyafter the operation of the cylinder. In addition, in contrast to alinear increase in the capacity of the cylinder due to the cylinderoperation, the fluid entry exhibits an asymptotic increase, thus posinga primary lag. Moreover, the suction quantity does not become identicalcompletely even after a sufficient time elapsed after cylinderoperation, thus posing an offset. This is applicable also to theresponse upon the fluid discharge.

Accordingly, the optimum parameter determining part determines, based onthe response property of the fluid which is suctioned into or dischargedfrom the column tip, the optimum parameters when taking the resistanceinto consideration based on this FIG. 9( b). Thus, by defining a suctioncorrected quantity or a discharge corrected quantity involving theoffset fluid quantity as an optimum parameter for a desired suctionquantity or discharge quantity, the influence by the offset can bereduced, and when the resistance in the column tip is high then thedetermination is made so that the wasted time and the primary lag arereduced by suctioning slowly. In addition, by defining the time untilstability after the fluid is suctioned into or discharged from thecolumn after completion of the cylinder operation as “waiting time aftersuction” and “waiting time after discharge”, the starting time for thenext operation is determined as an optimum parameter.

The optimum parameter includes a “stirring volume”. The stirring volumemay vary depending on the quantity of a test sample. Depending on theproportion between the packing volume and the valid column regioncapacity, the quantity of the liquid capable of being suctioned mayvary. When the packing volume is enclosed at 100% in the valid columnregion, the subject fluid can be introduced in a stirring volumeexceeding this valid column region, but at a lower % it should berequired that the stirring volume of the subject fluid is determined tobe a value not exceeding the valid column region capacity for thepurpose of preventing air from coming into.

In this manner, while for each structural requirement and processingrequirement the respective optimum parameters should be determined, sucha case-by-case determination of the optimum parameters is quitetiresome. Accordingly, in this embodiment, from standard structuralrequirement and processing requirement, standard optimum parameters aredetermined preliminarily, and in other cases the optimum parameters areestimated based on the ratio to or difference from the standard factors.

Accordingly, the optimum parameter determining part determines theoptimum parameters using the column tip 78 shown in FIG. 9( a) as astandard. In such a case, the standard optimum parameters are determinedfor the stirring volume to be 800μ liter in this case, for the stirringcycle to be 10 times in this case, for the suction flow rate to be 70μliter/second in this case, for the waiting time after suction to be 5seconds in this case, for the discharge flow rate to be 70μ liter/secondin this case, for the waiting time after discharge to be 5 seconds inthis case. The suction flow rate and the discharge flow rate here arecylinder-driven flow rates and are not the flow rates of the fluidsuctioned or discharged actually.

Otherwise, respective optimum parameters are calculated considering theratio to the standard value to the inner diameter of the resistance ofthe packing, the ratio to the standard value for the valid columnlength, and the ratio to the particle size, the optimum parameters arechanged considering their tendencies.

FIG. 10 shows an example of the determination of the optimum parametersother than the standard optimum parameters.

FIG. 10( a) indicates that when compared to the response of a fluid tothe packing 68 having a standard particle size d of 90 μm (long-dottedline) one having the particle size d of 34 μm (otherwise identicalfactors) leads to a higher plugging density which leads to a higherresistance, resulting in an increase in the “wasted time”, as well as afurther increase in the primary lag, and also an increase in the offsetlevel. Accordingly, as shown in FIG. 10( b), in order to achieve, evenwhen the particle size is 34 μm, the flow rate of the fluid when theparticle size is 90 μm, the cylinder operation rate should be increase.Therefore, in this case, the optimum parameters are determined to effecteither or all of the followings: (1) the waiting time after suction anddischarge is prolonged; (2) The stirring volume is increased; (3) Forachieving an actual suction and discharge flow rate of the fluid whichis identical to that with the mean particle size of 90 μm, the suctionand discharge flow rate is set at a higher rate.

Based on FIG. 11, a column tip processing method according to the fourthembodiment of the invention is described.

To process a column tip, first, in Step S1, the column tip 78 isinitialized. Upon initialization of the column tip 12, the column tip 12employed and the processing thereof are designated from the operationpanel 24 and the lid-fitted IC card insertion port 30 as the designatingpart. Thus, the number of test samples to be processed, the quantity ofthe test sample liquid, the positions where the test samples are placed,the processing protocol, the column regeneration processing if any areentered. The processing protocol includes the details of the processingcontents as well as the control parameter set thereof.

T Step S1 column initialization step involves, as a preliminaryprocedure, removal of the upper lid of the column tip 12 shown in FIG. 4while housing 12 tips in the 12 tip housing parts 61 in a tip rack 18 ofthe housing part group 20. The nozzle head 15 is moved relatively in thedirection of the X axis to this tip housing part 61, this nozzle head 15is lowered in the direction of the Z axis, and the nozzles 14 areinserted and put all at once to the opening 71 for fitting, wherebyfitting 12 column tips 12 to the nozzle head 15.

Subsequently in Step S11, the nozzle head 15 is raised in the directionof the Z axis, the column tips 12 are moved along the direction of the Xaxis, and the cap 72 is allowed to be placed under the detaching plate42 in this tip rack, whereby inserting so that the tube having a smalldiameter 66 reaches the holes 59 a. Then, the nozzle head 15 is raisedin the direction of the Z axis to drawn the cap 72 down apart.

In Step S12, the activation maintaining fluid housed in the column tips12 is allowed to be discharged into a waste tank (not shown), and thenin Step S13 the X axis moving body 94 is moved along the direction ofthe X axis, whereby moving the nozzle head 15 relatively to the reagentrack 55 as the fluid housing part group. Then, after washing andactivation are effected, the processing for conditioning the packing isconducted. This is accomplished in such a manner that a certain buffersolution as a subject fluid is suctioned a discharged repetitively(equilibration). In such a case, as suction and discharge parameters,the buffer position, the suction position, the stirring fluid volume,the number of stirring cycles, the suction flow rate, the halting timeafter suction, the discharge flow rate, and the halting time afterdischarge are determined.

In Step S14, a discharge motion is effected for discharging the washingand activation fluid thoroughly from the column tips. In such a case, asparameters, the height which prevents the washing or activation fluidfrom being brought into contact with the outside of the tip as far aspossible, the start waiting time, the discharge quantity, the dischargeflow rate are determined.

The sample adsorption step in Step S2 involves Step S21 in which thenozzle head 15 is moved to the position of the sample tube 60 on thestage 13 on which the sample as a subject fluid is housed, whereuponinserting the hollow end 69 of the column tip 12 into this sample tube60. In Step S22, by repeating suction and discharge, the packing and thesubject fluid are brought into contact with each other, whereby allowingthe biological substance to be adsorbed onto the packing. Upon this, thecontrolling part determines the optimum parameters as mentioned above,and then, based on these optimum parameters, controls the suction anddischarge mechanism and the moving means.

As suction and discharge parameters, “stirring volume”, “the number ofstirring cycles and time period”, “suction flow rate”, “halting timeafter suction”, “discharge flow rate”, “halting time after discharge”are exemplified. For these parameters, the optimum parameters aredetermined taking the followings into consideration. The “stirringvolume” is varied depending on the quantity of a test sample. Dependingon the packing and the tip capacity encompassed by the structuralrequirement, the quantity of the fluid capable of being suctioned mayvary. With regard to the “number of stirring cycles and time period”,repetitive passage through the packing is required when the ability ofbinding to the ligand is poor. A prolonged contact time period isrequired. With regard to the “suction flow rate”, there is a risk ofpealing off unless the suction is slow enough when the ability ofbinding to the ligand is poor. When the ability of binding issufficient, it may sometimes be efficient to effect a rapid suctionwhereby establishing the suspending state of the packing. The “haltingtime after suction” is a time period during which the complete immersionof the packing in a test sample solution as a subject fluid ismaintained. Since the flow resistance becomes higher when the packing istoo dense or the packing retaining mesh is too fine, the fluid maysometimes not be suctioned completely unless a sufficient halting timeis provided. With regard to the “discharge flow rate”, there is a riskof pealing off unless the discharge is slow enough when the ability ofbinding to the ligand is poor. With regard to the “halting time afterdischarge”, since the flow resistance becomes higher when the packing istoo dense or the packing retaining mesh is too fine, the fluid maysometimes not be suctioned completely unless a sufficient halting timeis provided.

In Step S23, a processing is conducted for preventing any carryover ofunnecessary contaminants into the subsequent processing. Requiredparameters include “discharge height”, “waiting time for discharge”,“discharge quantity”, “discharge flow rate”, for which optimumparameters are determined taking the followings into account. The“discharge height” is a height which prevents the sample solution as asubject fluid from being brought into contact with the outside of thetip as far as possible. A height preventing any droplet from beingformed on the end when the tip is raised after the fluid discharged fromthe tip again is brought into contact with the sample fluid as a subjectfluid is designated. The “waiting time for discharge” designates a timeperiod until the unnecessary subject fluid present inside and outside ofthe tip drops down spontaneously to the lower end of the tip. The“discharge quantity” designates a quantity for complete discharge of theunnecessary fluid retained in the end of the tip. The “discharge flowrate” designates a flow rate for complete discharge of the unnecessaryfluid retained in the end of the tip.

Step S3 shows a washing step, including Step S31 for movement to thewashing position and Step S32 for stirring by allowing the washing fluidas a subject fluid to be suctioned into and discharged from the columntip 12 repetitively. As parameters in the washing step, those occurringby replacing the subject fluid from the sample in the sample adsorptionstep mentioned above into the washing solution are employed. Step S33 isa processing for preventing any carryover of unnecessary washing fluidinto the subsequent processing, and its parameters are those occurringby replacing the subject fluid in the parameters in Step S23 in thesample adsorption step mentioned above from the sample into the washingsolution. The washing step can be conducted repetitively until asufficient washing is achieved in a plurality of the fluid housing partsor in the fluid housing parts of different washing fluids.

Step S4 shows an eluting step, including Step S41 for movement to theposition which is the suction and discharge position as a parameter andwhere an eluted fluid is housed. The position where the eluted fluid ishoused is designated by ligand and the sample of the subject fluid. InStep S42, the column tip 12 is subjected to a repetitive suction anddischarge of the eluted fluid. When conducting concentration, the elutedfluid may not discharge into the column sufficiently. In such a case,the stirring fluid quantity should be adjusted so that the eluted fluidis suctioned to the position allowing all of the packing is passedthrough.

In Step S43, the entire quantity of the eluted fluid is suctioned.Accordingly, as a suction quantity, the parameters are adjusted so thatan extra quantity in addition to the quantity of the eluted fluid issuctioned.

In Step S44, the nozzle head 15 is moved relatively to the recoveryvessel position. Step S45 serves to discharge the entire quantity. StepS46 serves to discharge an extra quantity in addition to the elutedfluid flowing quantity, since the eluted fluid tends to remain in thecolumn tip 12. Before conducting the extra discharge, a waiting time forthe fluid in the column tip 12 to drip down to the end of the tip isprovided.

Step S5 is a follow-up processing. This step conducts a processing for afuture use when a used column tip 12 can be used again.

Step S51 conducts a regenerating processing. The regenerating processingconducts the packing washing and the suction and discharge of theregenerating fluid for establishing a state suitable for the storage.Accordingly, stirring quantity, the number of stirring cycles, stirringrate are selected. In Step S52, a storage fluid or an activationmaintaining fluid is suctioned. The suction of the storage fluid servesto fill the storage fluid for preventing the packing in the column frombeing dried or fungal organisms from being allowed to grow.

In Step S53, for removing the column tip 12 from the nozzle 14 whileallowing the storage fluid to remain still as being filled, the end isfitted with a cap. In the case of a high throughput system, the fittingis accomplished automatically. In the case of a compact-type system fora small number of samples, the fitting may be accomplished manuallythrough a user interface.

FIGS. 12 to 14 show specific processings (processings according to thefourth embodiment to the sixth embodiment) with regard to anycombination of one or more types of column tips, filter tips ordispensing tips (at least one type of columns should be included).

FIG. 12 shows an example of a recombinant protein purificationprocessing, including a magnetic particle-based fusion proteinpurification using the dispensing tips and a protein analysispretreatment using gel filtration column tips. In Steps S7-1 and S7-2,the dispensing tips 106 are fitted to the nozzles 14 of the nozzle head15, and then, using these dispensing tips 106, a subject fluidcontaining a GST fusion protein or histidine-tagged fusion protein and amagnetic particle suspension having an anti-GST antibody or glutathioneand a nickel ion immobilized thereon are reacted with each other bystirring.

Step S7-3 serves to use a magnetic means 32 provided in the nozzle headto allow the magnetic particles to be adsorbed onto the inner wall ofthe dispensing tip 106 whereby accomplishing separation. Step S7-4serves to wash with a washing fluid. Step S7-5 serves to elute thefusion protein from the magnetic particles using a salt solution at ahigh concentration or a solution of a reduced form of glutathione in thecase of the GST fusion protein and an imidazole solution in the case ofthe histidine-tagged fusion protein, followed by housing in tubes or thelike on the stage 13.

Then, in Step S7-6 the dispensing tips are detached from the nozzle 14.Then, in Step S7-9, the gel filtration column tips 96 such as ofSephadex or Sephacryl are housed in the housing part group 20. In StepS7-10, the column tips 96 are made free of the storage fluid formaintaining activation, and equilibrated. In Step S7-11, the dispensingtips are used to add the eluted fluid to these gel filtration columntips 96, and then, after connection to the nozzles 14 of the nozzle head15, a high molecular weight fractionation (protein) is conducted in StepS7-12 to recover the proteins sequentially in the order of highermolecular weights, followed by desalting or removal of low molecularweight contaminants, whereby accomplishing purification.

FIG. 13 shows an example of a processing for removing major proteins ina serum, which employs an affinity column tip in combination with adispensing tip.

In Step S8-1, the column tips 78 each enclosed with an affinity gelhaving antibodies (several types) specific to the major proteinsimmobilized thereon are used as being fitted to the nozzles 14. In StepS8-2, the column tips are equilibrated. In Step S8-3, the serum issuctioned and discharged to allow the major proteins to be adsorbed. InStep S8-4, a serum now free of the major proteins is discharged.

A protein extracted by a two dimensional electrophoresis is subjected toStep S8-7 where after the column tips are detached and then thedispensing tips are fitted, and the trypsin enzyme bound to the magneticparticles is used to effect an enzymatic digestion, followed by usingthe magnetic force means to allow the magnetic particles to be adsorbedonto the inner wall of these dispensing tips whereby effectingseparation, followed by Step S8-8 where a peptide sample for a massspectrometry is obtained.

FIG. 14 shows an example of an antibody purification processing,especially a pretreatment of a protein analysis sample using an affinitycolumn tip, a gel filtration column tip, and a filter tip.

In Step S9-1, a column tip 78 enclosed with an affinity gel such asProtein A or Protein G is fitted to the nozzle 14. In Step S9-2, thiscolumn tip 78 is equilibrated. In Step S9-3, a mouse ascites issuctioned and discharged repetitively to allow the antibody to beadsorbed. In Step S9-4, the column tip 78 is washed. In Step S9-5, theantibody adsorbed onto the packing is eluted with a glycine HCl buffersolution. The eluted fluid is discharged to and housed in an appropriatefluid housing part in the housing part group 20.

After detaching the column tip 78 from the nozzle 14 of the nozzle head15, the column tip 96 for the gel filtration is housed in the tip rackin the housing part group 20 in Step S9-10. In Step S9-11, the columntip 96 for the gel filtration is equilibrated. In Step S9-12, thedispensing tip is fitted to the nozzle head and the fluid eluted fromthe opening 71 for fitting is added to the column tip 96 for the gelfiltration. After detaching the dispensing tip from the nozzle 14 of thenozzle head 15, the column tip 96 for this gel filtration is fitted toeach nozzle 14 of the nozzle head 15, and then discharge in Step S9-13followed by a high molecular weight fractionation results in recoveryand purification of the protein.

In Step S9-14, after detaching the column tip 96 from the nozzle 14, theproduct is inserted to a filter tip 102 housed in the housing part ofthe stage 13, and then discharged and concentrated by fitting thisfilter tip 102 to the nozzle 14 followed by applying a pressure by thesuction and discharge mechanism.

FIG. 15 shows a buffer solution as a subject fluid employed whenconducting elution of an intended biological substance adsorbed in acolumn tip processing device 10 according to the 8th embodiment of theinvention.

In this embodiment, a column tip 78 is employed, and as a packing asubstance employed in an affinity chromatography or an ion exchangechromatography may for example be enclosed, and allowed to adsorb theintended protein, such as a bacteria-derived enzyme. In order to elutethe adsorbed intended protein, buffer solutions having a certaindifference from each other in the concentration of a salt along with theconcentration gradient are housed in one reagent prepack cartridge 58present on the state 13 of the column tip processing device 10. Here,the difference in the concentration of the salt is not necessarilyconstant. In this case, as shown in FIG. 15, those having theconcentrations starting from 0 mM for washing and thereafter, by thedifference in the concentration of 50 mM, from 50 mM to 400 mM (E1 toE8) and 600 mM (E9) are housed. These buffer solutions can be producedby mixing the buffer solution (R) at 1000 mM and the solution (H) at 0mM housed similarly in this prepack using the dispensing tip fitted tothe nozzle 14. For example, the buffer solution at 500 mM can readily beproduced by mixing R with H each in 50 percents.

Each embodiment thus described is presented specifically for the purposeof a better understanding of the present invention, and is not intendedto impose any restriction on other embodiments. Accordingly, variationscan be made as far as the subject matter of the invention is notchanged. For example, the embodiments of the invention described abovecan be applied to a processing in the field of genetics, although theyare described mainly with regard only to proteins. For example, bycombining a dispensing tip employing a magnetic particle with a gelfiltration column tip, it is possible to conduct a nucleic acidextraction, a PCR product purification and capture of intendedsubstances, as well as a concentrating processing. In addition, bycombining a dispensing tip employing a magnetic particle with a filtertip, it is possible to conduct a nucleic acid extraction, a PCR productpurification, concentration or a high purity purification (completeremoval of a trace amount of magnetic particles).

Also in the field of proteins, otherwise by using a dispensing tipemploying a magnetic particle and a filter tip, it can be used in afusion protein extraction and purification, a molecular sieve (highmolecular weight protein removal) processing. In addition, by using adispensing tip employing a magnetic particle, a gel filtration columntip and a dispensing tip of another type, it can be used for removal ofthe major protein, for exchange of a buffer solution, and for anenzymatic digestion (trypsin-immobilizing magnetic particle) processing.In addition, by using an affinity column tip and a gel filtration columntip, it can be used in a processing for removing the major proteins andexchanging buffer solutions, or in a processing for affinitypurification followed by buffer exchange. In addition, by using anaffinity column tip, a gel filtration column tip and a filter tip, itcan be used for removing the major proteins and exchanging buffersolutions, and then conducting a concentration processing. Moreover, byusing an affinity column tip, a gel filtration column tip, a filter tipand a dispensing tip, it can be used in a major protein removingprocessing, a buffer exchange, concentration and enzymatic digestionprocessings.

INDUSTRIAL APPLICABILITY

The present invention relates to a column tip processing device and acolumn tip processing method. The invention relates to various fieldsrequiring a handling of biological high molecular weight substances andbiological low molecular weight substances including

genetics, immunology, amino acids, proteins, saccharides and the like,such as industrial fields, agricultural field including foods,agricultures, marine resource processings, pharmaceutical fields,medical fields including hygiene, welfare, immunology, disease, geneticsand the like, chemical or biological or physical fields and the like.The invention is a method which is effective especially in a case wherea large number of reagents or substances are employed in a series ofprocessings which is conducted in a certain order continuously.

DESCRIPTION OF REFERENCE NUMERALS

-   10: Column tip processing device-   12, 78, 82, 96: Column tip-   14: Nozzle-   15: Nozzle head-   16: Suction and discharge mechanism-   20: Housing part group-   24: Control part

1. A column tip processing device comprising: a nozzle head having asingle or multiple-channeled nozzle; a suction and discharge mechanismconducting suction or discharge of a gas via the nozzle; one or moretypes of column tips each having a tip-shaped vessel having a fittingopening to be fitted to the nozzle and a hollow end through which afluid can flow in or out in response to the suction or discharge of thegas and a packing enclosed in the tip-shaped vessel; a stage providedwith a fluid housing part group including a plurality of fluid housingparts into each of which the hollow end can be inserted and which houseor can house various solutions; and, moving means for moving the nozzlehead relatively to the fluid housing part group; and also having acontrolling part which controls the suction and discharge mechanism andthe moving means with regard to quantities, pressure, flow rate, thenumber of cycles, time or position of the suction or discharge by thenozzle based on a structural requirement relating to the structure ofthe one or more types of column tips to be fitted to the nozzle and aprocessing requirement relating to the processing contents involving oneor more types of subject fluids subjected to the suction or discharge ofthe column tips, wherein the controlling part has a designating partwhich designates the column tips and a processing using the column tips,a requirement generating part which generates a structural requirementrelating to the designated column tips and a processing requirementrelating to the designated processing, and an optimum parameterdetermining part which determines, based on the generated structuralrequirement and processing requirement, an optimum parameter with whichthe suction and discharge mechanism and the moving means should be inaccordance, wherein the optimum parameter determining part determines anoptimum parameter relating to suction or discharge, based on thegenerated corresponding structural requirement and processingrequirement for each of the column tips and processing contents, inorder to reduce the difference in time between the suction or dischargeoperation starting time for the suction and discharge mechanism and thefluid movement starting time for column tips and also the offset ofsuction or discharge operation quantity and the suction or dischargequantity of the fluid for the column tip after achieving the operationquantity.
 2. The column tip processing device according to claim 1,wherein the packing is enclosed in the tip-shaped vessel using at leastone filter provided in a manner to partition the tip-shaped vessel, thestructural requirement includes a plurality of items relating to thestructure of the tip-shaped vessel, the structure of the filter or themorphology, type and nature of the packing enclosed, and the processingrequirement includes a plurality of items relating to the processingcontents including each housing position, type, nature or quantity ofone or more types of the subject fluids subjected to the suction ordischarge of the column tips.
 3. (canceled)
 4. (canceled)
 5. The columntip processing device according to claim 1, wherein the optimumparameter determining means determines, based on a standard structuralrequirement which sets predetermined one or two standard column tips andat least a part of the plurality of the items of the structuralrequirement corresponding to the standard processing contents at one ormore standard values and a standard processing requirement which sets atleast a part of the plurality of the items of the processing requirementat one or more standard values, an optimum parameter corresponding tostructural requirements and processing requirements other than thestandard structural requirements and the standard processingrequirement.
 6. The column tip processing device according to claim 1,wherein the stage has a temperature raising and lowering vessel whichraises or lowers the temperature in response to an external signal, atleast one of the fluid housing part is housed in the temperature raisingand lowering vessel, and the control of the temperature of the subjectfluid is conducted on the moving means based on the processingrequirement.
 7. The column tip processing device according to claim 1,wherein on the stage one or more said column tips, one filter tip havinga fitting opening to be fitted to a nozzle, or one dispensing tip, and adetaching part for detaching the column tip, filter tip or dispensingtip fitted to the nozzle are provided, the controlling part conducts, onthe suction and discharge mechanism and the moving means, the control ofthe fitting and detachment of the column tip, filter tip or dispensingtip based on the structural requirement and the processing requirement.8. The column tip processing device according to claim 1, wherein atleast a part of the fluid housing part group is provided with apiercable thin film covering the opening of the fluid housing part, thenozzle head is provided with a piercing pin capable of piercing the thinfilm, and the control of the thin film piercing is conducted on themoving means based on the processing requirement.
 9. The column tipprocessing device according to claim 1, wherein the nozzle head isprovided with a fall off preventing part which prevents the fall offfrom the nozzle by engaging with the tip-shaped vessel of the column tipfitted to the nozzle, and the control of the fall off prevention and acancellation thereof by the fall off preventing part is conducted on themoving means based on the structural requirement and the processingrequirement.
 10. A column tip processing method comprising: based on astructural requirement of one or more types of column tips each having atip-shaped vessel having a fitting opening conducting suction ordischarge of a gas by a suction and discharge mechanism and a hollow endthrough which a fluid can flow in or out in response to the suction ordischarge of the gas and a packing enclosed in the tip-shaped vessel,and a processing requirement relating to the processing contentsinvolving one or more types of subject fluids subjected to the suctionor discharge of the column tips, a fitting step for fitting a column tipto the nozzle at the fitting opening by moving the nozzle relativelybetween it and the housing parts of the column tip housed therein; acontacting step for inserting the hollow end into the fluid housing partby a relative movement between one or more fluid housing part housingthe subject fluid and the nozzle whereby suctioning and discharging thesubject fluid according to quantities, pressure, flow rate, the numberof cycles, time or position of the suction or discharge by the nozzledetermined based on the requirement, whereby bringing the packing intocontact with the subject fluid; a discharging step for discharging thesubject fluid from the column tip into one or more the fluid housingparts; a designating step for designating column tips to be fitted andprocessing contents to be processed; and, a generating step forgenerating a structural requirement corresponding to the column tipsdesignated and generating a processing requirement corresponding to theprocessing contents designated; wherein the contacting step has anoptimum parameter determining step for determining an optimum parameterbased on the generated structural requirement and processingrequirement, and a contact executing step for moving the nozzle whileexecuting suction or discharge, wherein the optimum parameterdetermining step determines suction or discharge parameters, based onthe generated corresponding structural requirement and processingrequirement for each of the column tips and processing contents, whileconsidering the difference in time between the suction or dischargeoperation starting time for the suction and discharge mechanism and thefluid movement starting time for column tips, the offset of suction ordischarge operation quantity and the suction or discharge quantity ofthe fluid for the column tip after achieving the operation quantity, andthe details of the reaction of the packing with substances contained inthe subject fluid, in order to correct the difference between thetargeted suction or discharge quantity at the completion of the suctionor discharge and the suction or discharge quantity of the fluid for thecolumn tip after completion of the operation.
 11. The column tipprocessing method according to claim 10, wherein the packing is enclosedin the tip-shaped vessel using at least one filter provided in a mannerto partition the tip-shaped vessel, the structural requirement includesa plurality of items relating to the structure of the member or thetip-shaped vessel fitted to the nozzle, the structure of the filter orthe morphology, type and nature of the packing enclosed, and theprocessing requirement includes a plurality of items relating to theprocessing contents including each housing position, type, nature orquantity of one or more types of the subject fluids subjected to thesuction or discharge of the column tips.
 12. (canceled)
 13. (canceled)14. The column tip processing method according to claim 10, wherein theoptimum parameter determining step determines, based on a standardstructural requirement for which one or more standard values are set forat least a part of the plurality of the items of the structuralrequirement generated corresponding to the predetermined standard columntips and the standard processing contents and a standard processingrequirement for which one or more standard values are set for at least apart of the plurality of the items of the processing requirement, anoptimum parameter corresponding to structural requirements andprocessing requirements other than the standard structural requirementsand the standard processing requirement for those other than thepredetermined standard column tips and processing contents.
 15. Thecolumn tip processing method according to claim 10, comprising, afterthe discharge step, a step for washing the packing enclosed in thecolumn tip by allowing a washing fluid as a subject fluid to besuctioned into and discharged from the column tip and an elution stepfor introducing an eluent into the column tip whereby eluting theprocessing subject fluid-carried biological substances which wereadsorbed to, captured by and reacted with or bound to the packing. 16.The column tip processing method according to claim 11, wherein, in thecontact step, a temperature raising and lowering step for raising andlowering the temperature of the subject fluid based on the processingrequirement is provided, and the raising and lowering of the temperatureis conducted by means of a relative movement between the nozzle and thetemperature raising and lowering vessel provided on the stage.
 17. Thecolumn tip processing method according to claim 10, further comprising adetachment step for detaching the column tips fitted to the nozzle,wherein the detaching step is conducted by means of a relative movementbetween the detaching part provided on the stage and the nozzle based onthe structural requirement and processing requirement.
 18. The columntip processing method according to claim 10, wherein, after thedetaching step, fitting of at least one said column tip of another type,one filter tip connectable to the nozzle, or a dispensing tip housed onthe stage is conducted, based on the structural requirement andprocessing requirement, by means of a relative movement between thenozzle and the housing part in which the column tip, filter tip ordispensing tip is housed.
 19. An optimum parameter generating program,for a column tip processing device comprising: a nozzle head having asingle or multiple-channeled nozzle; a suction and discharge mechanismconducting suction or discharge of a gas via the nozzle; one or moretypes of column tips each having a tip-shaped vessel having a fittingopening to be fitted to the nozzle and a hollow end through which afluid can flow in or out in response to the suction or discharge of thegas and a packing enclosed in the tip-shaped vessel; a stage providedwith a fluid housing part group into which the hollow end can beinserted and which houses or can house various solutions; and, movingmeans for moving the nozzle head relatively to the fluid housing partgroup, wherein the optimum parameter generating program incorporates oneor more column tips fitted to the nozzle and a designating data whichdesignates the processing conducted using the column tips, generates,based on the designating data, a structural requirement data relating tocorresponding column tip structure and a processing requirement datarelating to the processing contents involving one or more types ofsubject fluids subjected to the suction or discharge of the column tipsincluded in corresponding processing, and determines, based on therequirements generated, optimum parameter data prescribing quantities,pressure, flow rate, the number of cycles, time or position of thesuction or discharge by the nozzle for the suction and dischargemechanism and the moving means, wherein the optimum parameter decisiondetermines an optimum parameter data relating to suction or discharge,based on the generated corresponding structural requirement data andprocessing requirement data for each of the column tips and processingcontents, while considering the difference in time between the suctionor discharge operation starting time for the suction and dischargemechanism and the fluid movement starting time for column tips, theoffset of suction or discharge operation quantity and the suction ordischarge quantity of the fluid for the column tip after achieving theoperation quantity, and the details of the reaction of the packing withsubstances contained in the subject fluid, while considering the suctionor discharge parameter and the difference in time between the suction ordischarge operation starting time and the fluid movement starting timefor column tips, the offset of suction or discharge operation quantityand the suction or discharge quantity of the fluid for the column tipafter achieving the operation quantity, and the details of the reactionof the packing with substances contained in the subject fluid.
 20. Theoptimum parameter generating program according to claim 19, wherein thepacking is enclosed in the tip-shaped vessel using at least one filterprovided in a manner to partition the tip-shaped vessel, the structuralrequirement data include a plurality of items relating to the structureof the member or the tip-shaped vessel to be fitted to the nozzle, thestructure of the filter or the morphology or nature of the packingenclosed, and the processing requirement data include a plurality ofitems relating to the processing contents including each housingposition, nature or quantity of one or more types of the subject fluidssubjected to the suction or discharge of the column tips.
 21. (canceled)22. (canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled) 26.(canceled)
 27. (canceled)
 28. (canceled)
 29. (canceled)
 30. (canceled)31. (canceled)
 32. (canceled)
 33. The column tip processing methodaccording to claim 14, wherein, in the contact step, a temperatureraising and lowering step for raising and lowering the temperature ofthe subject fluid based on the processing requirement is provided, andthe raising and lowering of the temperature is conducted by means of arelative movement between the nozzle and the temperature raising andlowering vessel provided on the stage.
 34. The column tip processingmethod according to claim 15, wherein, in the contact step, atemperature raising and lowering step for raising and lowering thetemperature of the subject fluid based on the processing requirement isprovided, and the raising and lowering of the temperature is conductedby means of a relative movement between the nozzle and the temperatureraising and lowering vessel provided on the stage.